The mechanisms of cholesterol transfer between cells and lipoproteins are central to our understanding of cholesterol accumulation in vascular wall cells during atherosclerosis development. The formation of lipid-laden foam cells within the arterial intima is a hallmark of atheroclerotic disease. Previous studies showed that free cholesterol (FC) is transferred between cells and lipoproteins by an aqueous diffusion pathway and by an apolipoprotein-dependent microsolubilization of membrane FC and phospholipid (PL). Recent studies have identified a novel FC transfer pathway that involves the cell surface receptor, scavenger receptor BI (SR-BI). The SR-BI-dependent pathway greatly accelerates the flux of FC between cells and lipoproteins and may play key roles in cholesteryl ester (CE) accumulation and FC efflux from vascular cells and in the transfer of CE and FC from lipoproteins to the liver. This proposal is focused on the mechanisms by which SR-BI alters cellular cholesterol metabolism. Aim 1 will employ domain swapping and mutagenesis approaches to identify SR-BI domains responsible for the effects of SR-BI on FC efflux, changes in plasma membrane lipid composition and organization, and HDL binding. Aim 2 will develop cell culture models in which SR-BI expression is under control of a tetracycline-regulated promoter to permit tightly regulated expression of SR-BI in COS7 cells and in the J774 macrophage model foam cell. Aim 3 will investigate the relationships between SR-BI expression and the intracellular metabolism of cholesterol in cells with inducible SR-BI expression and in macrophage derived from wild type and SR-BI-deficient mice. These experiments will test the effects of SR-BI on FC and CE accumulation from various lipoproteins and distinguish between SR-BI-mediated effects on cholesterol uptake from those on CE hydrolysis and FC efflux from the cell. PL- enriched HDL will be used to test the hypothesis that large PL-enriched HDL are more efficient acceptors for SR-BI-mediated FC efflux as compared to PL-depleted HDL. The effects of SR-BI on lipid domains of the plasma membrane will be evaluated by determinations of the PL, FC, and sphingomyelin composition of plasma membrane and membrane subfractions prepared by detergent based and non-detergent based protocols. These experiments will provide new information that is fundamental to our understanding of SR-BI function, its role in the regulation of cellular cholesterol metabolism, and its impact on the formation of lipid-laden foam cells in the vascular wall.